Ginsenoside Rb2 exhibits therapeutic value for male osteoporosis in orchiectomy mice by suppressing osteoclastogenesis and modulating NF-κB/MAPK signaling pathways.

Osteoporosis (OP) is a systemic disorder characterized by decreased bone mass as well as deteriorated microarchitecture. Although OP in men is common, it has received much less attention than that in women. Ginseng, a famous traditional herb in Asia, is used to strengthen and repair bones by invigorating vital bioenergy and maintaining body homeostasis in dietary intake and clinical applications. However, there is currently no study investigating the impact of ginseng and its active compounds on male osteoporosis. In this study, RNA sequencing and bioinformatic analysis were conducted to reveal the influence of Ginsenoside-Rb2 on RAW264.7 cells and its underlying signaling pathways. The potential anti-osteoporosis effects of Rb2 as well as its molecular mechanisms were elucidated in RAW264.7 cells and BMMs by TRAP staining, F-actin belt staining, qRT-PCR and WB. Moreover, orchiectomy (ORX) was utilized to demonstrate the influence of Rb2 on bone mass loss in vivo by micro-CT scanning, and H&E, TRAP, and IHC staining. The results suggested that Rb2 suppressed osteoclastogenesis and mitigated bone loss in orchiectomy mice through NF-κB/MAPK signaling pathways. These findings indicate that ginseng as well as its active component Rb2 have potential therapeutic value in the management of osteoporosis in men.

Syringic acid promotes cartilage extracellular matrix generation and attenuates osteoarthritic cartilage degradation by activating TGF-ß/Smad and inhibiting NF-κB signaling pathway.

Osteoarthritis (OA) is a common chronic degenerative disease which is characterized by the disruption of articular cartilage. Syringic acid (SA) is a phenolic compound with anti-inflammatory, antioxidant, and other effects including promoting osteogenesis. However, the effect of SA on OA has not yet been reported. Therefore, the purpose of our study was to investigate the effect and mechanism of SA on OA in a mouse model of medial meniscal destabilization. The expressions of genes were evaluated by qPCR or western blot or immunofluorescence. RNA-seq analysis was performed to examine gene transcription alterations in chondrocytes treated with SA. The effect of SA on OA was evaluated using destabilization of the medial meniscus model of mice. We found that SA had no obvious toxic effect on chondrocytes, while promoting the expressions of chondrogenesis-related marker genes. The results of RNA-seq analysis showed that extracellular matrix-receptor interaction and transforming growth factor-ß (TGF-ß) signaling pathways were enriched among the up-regulated genes by SA. Mechanistically, we demonstrated that SA transcriptionally activated Smad3. In addition, we found that SA inhibited the overproduction of lipopolysaccharide-induced inflammation-related cytokines including tumor necrosis factor-α and interleukin-1ß, as well as matrix metalloproteinase 3 and matrix metalloproteinase 13. The cell apoptosis and nuclear factor-kappa B (NF-κB) signaling were also inhibited by SA treatment. Most importantly, SA attenuated cartilage degradation in a mouse OA model. Taken together, our study demonstrated that SA could alleviate cartilage degradation in OA by activating the TGF-ß/Smad and inhibiting NF-κB signaling pathway.

Effect of solar proton events on test mass for gravitational wave detection in the 24th solar cycle.

Free-falling cubic Test Masses (TMs) are a key component of the interferometer used for low-frequency gravitational wave (GW) detection in space. However, exposure to energetic particles in the environment can lead to electrostatic charging of the TM, resulting in additional electrostatic and Lorentz forces that can impact GW detection sensitivity. To evaluate this effect, the high-energy proton data set of the Geostationary Operational Environmental Satellite (GOES) program was used to analyze TM charging due to Solar Proton Events (SPEs) in the 24th solar cycle. Using the Geant4 Monte Carlo toolkit, the TM charging process is simulated in a space environment for SPEs falling into three ranges of proton flux: (1) greater than 10 pfu and less than 100 pfu, (2) greater than 100 pfu and less than 1000 pfu, and (3) greater than 1000 pfu. It is found that SPEs charging can reach the threshold within 535 s to 18.6 h, considering a reasonable discharge threshold of LISA and Taiji. We demonstrate that while there is a somewhat linear correlation between the net charging rate of the TM and the integrated flux of [Formula: see text] 10 MeV SPEs, there are many cases in which the integrated flux is significantly different from the charging rate. Therefore, we investigate the difference between the integral flux and the charging rate of SPEs using the charging efficiency assessment method. Our results indicate that the energy spectrum structure of SPEs is the most important factor influencing the charging rate. Lastly, we evaluate the charging probability of SPEs in the 24th solar cycle and find that the frequency and charging risk of SPEs are highest in the 3rd, 4th, 5th, 6th, and 7th years, which can serve as a reference for future GW detection spacecraft.

Isoimperatorin attenuates bone loss by inhibiting the binding of RANKL to RANK.

Osteoporosis, an immune disease characterized by bone mass loss and microstructure destruction, is often seen in postmenopausal women. Isoimperatorin (ISO), a bioactive, natural furanocoumarin isolated from many traditional Chinese herbal medicines, has therapeutic effects against various diseases; however, its effect on bone homeostasis remains unclear. In this study, we investigated the effect of ISO on the differentiation and activation of osteoclast and its molecular mechanism in vitro, and evaluated the effect of ISO on bone metabolism by ovariectomized (OVX) rat model. In vitro experiments showed that ISO affected RANKL-induced MAPK, NFAT, NFATc1 trafficking and expression, osteoclast F-actin banding, osteoclast-characteristic gene expression, ROS inhibitory activity, and calcium oscillations, NF-κB signaling pathway. In vivo experiments showed that oral administration of ISO effectively reduced bone loss caused by ovariectomy and retained bone mass.Collectively, ISO inhibits RANK/RANKL binding, thereby reducing the activity of NFATc1, calcium, and ROS and inhibiting osteoclast generation. In addition, ISO protects bone mass by slowing osteoclast production and downregulating NFATc1 gene and protein expression in the bone tissue microenvironment and inhibits OVX-induced bone loss in vivo.

Ginsenoside Compound K Enhances Fracture Healing via Promoting Osteogenesis and Angiogenesis.

Fractures have an extraordinarily negative impact on an individual's quality of life and functional status, particularly delayed or non-union fractures. Osteogenesis and angiogenesis are closely related to bone growth and regeneration, and bone modeling and remodeling. Recently Chinese medicine has been extensively studied to promote osteogenic differentiation in MSCs. Studies have found that Ginseng can be used as an alternative for tissue regeneration and engineering. Ginseng is a commonly used herbal medicine in clinical practice, and one of its components, Ginsenoside Compound K (CK), has received much attention. Evidence indicates that CK has health-promoting effects in inflammation, atherosclerosis, diabetics, aging, etc. But relatively little is known about its effect on bone regeneration and the underlying cellular and molecular mechanisms. In this study, CK was found to promote osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) by RT-PCR and Alizarin Red S staining in vitro. Mechanistically, we found CK could promote osteogenesis through activating Wnt/ß-catenin signaling pathway by immunofluorescence staining and luciferase reporter assay. And we also showed that the tube formation capacity of human umbilical vein endothelial cells (HUVECs) was increased by CK. Furthermore, using the rat open femoral fracture model, we found that CK could improve fracture repair as demonstrated by Micro-CT, biomechanical and histology staining analysis. The formation of H type vessel in the fracture callus was also increased by CK. These findings provide a scientific basis for treating fractures with CK, which may expand its application in clinical practice.

Hydroxysafflor yellow A promotes osteogenesis and bone development via epigenetically regulating ß-catenin and prevents ovariectomy-induced bone loss.

In clinical treatment, there is increasingly prevalent that traditional Chinese medicine treats common bone diseases including osteoporosis. Hydroxysafflor yellow A (HSYA), one of the essential compounds of Safflower, has been used as the therapy for thrombus, myocardial ischemia, and inflammation, but its effect on osteogenesis through epigenetic control and ovariectomy-induced bone loss in vivo has not been explored. Therefore, the study aimed to explore the function and mechanism of HSYA on bone formation and development. We found HSYA could enhance the cell viability and promote osteogenesis of hBMSCs in vitro. Mechanistically, HSYA could increase the expression of ß-catenin leading to its accumulation in the nucleus and activation of downstream targets to promote osteogenesis. Besides, RNA-seq and quantitative RT-PCR and western blot showed KDM7A was significantly increased by HSYA. The occupancy of H3K27me2 on ß-catenin promoter was significantly decreased by HSYA, which could be reversed by silencing endogenous KDM7A. More importantly, HSYA promoted bone development in chick embryos and prevented ovariectomy (OVX)-induced bone loss in SD rats. Taken together, our study has shown convincing evidence that HSYA could promote osteogenesis and bone development via epigenetically regulating ß-catenin and prevent ovariectomy-induced bone loss.

Carnosol suppresses RANKL-induced osteoclastogenesis and attenuates titanium particles-induced osteolysis.

Osteolysis is a common medical condition characterized by excessive activity of osteoclasts and bone resorption, leading to severe poor quality of life. It is essential to identify the medications that can effectively suppress the excessive differentiation and function of osteoclasts to prevent and reduce the osteolytic conditions. It has been reported that Carnosol (Car), isolated from rosemary and salvia, has anti-inflammatory, antioxidative, and anticancer effects, but its activity on osteolysis has not been determined. In this study, we found that Car has a strong inhibitory effect on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation dose-dependently without any observable cytotoxicity. Moreover, Car can inhibit the RANKL-induced osteoclastogenesis and resorptive function via suppressing NFATc1, which is a result of affecting MAPK, NF-κB and Ca2+ signaling pathways. Moreover, the particle-induced osteolysis mouse model confirmed that Car could be effective for the treatment of bone loss in vivo. Taken together, by suppressing the formation and function of RANKL-induced osteoclast, Car, may be a therapeutic supplementary in the prevention or the treatment of osteolysis.

Catharmus tinctorius volatile oil promote the migration of mesenchymal stem cells via ROCK2/Myosin light chain signaling.

MSC transplantation has been explored as a new clinical approach to stem cell-based therapies for bone diseases in regenerative medicine due to their osteogenic capability. However, only a small population of implanted MSC could successfully reach the injured areas. Therefore, enhancing MSC migration could be a beneficial strategy to improve the therapeutic potential of cell transplantation. Catharmus tinctorius volatile oil (CTVO) was found to facilitate MSC migration. Further exploration of the underlying molecular mechanism participating in the pro-migratory ability may provide a novel strategy to improve MSC transplantation efficacy. This study indicated that CTVO promotes MSC migration through enhancing ROCK2 mRNA and protein expressions. MSC migration induced by CTVO was blunted by ROCK2 inhibitor, which also decreased myosin light chain (MLC) phosphorylation. Meanwhile, the siRNA for ROCK2 inhibited the effect of CTVO on MSC migration ability and attenuated MLC phosphorylation, suggesting that CTVO may promote BMSC migration via the ROCK2/MLC signaling. Taken together, this study indicates that C. tinctorius volatile oil could enhance MSC migration via ROCK2/MLC signaling in vitro. C. tinctorius volatile oil-targeted therapy could be a beneficial strategy to improve the therapeutic potential of cell transplantation for bone diseases in regenerative medicine.

MiR-539-5p negatively regulates migration of rMSCs induced by Bushen Huoxue decoction through targeting Wnt5a.

Bone fractures are very common, and above 5% of the fractures are impaired, leading to nonunions and severe disablilities. The traditional Chinese medicine Bushen Huoxue decoction (BHD) has been used to treat fracture in China. Our previous report has found that BHD promotes migration of rat mesenchymal stem cells (rMSCs) by activating Wnt5a signaling pathway. However, whether and how miRNAs are involved in modulating rMSCs migration induced by BHD has not been explored. In the present study, miRNA microarray analysis and further validation by real-time quantitative RT-PCR revealed that miR-539-5p was down-regulated in BHD-induced rMSCs. Transfection of miR-539-5p mimics suppressed rMSCs migration while the miR-539-5p inhibitor promoted rMSCs migration. Our results suggested that miR-539-5p was a negative regulator of migration of rMSCs induced by BHD. Target prediction analysis tools and Dual-luciferase reporter gene assay identified Wnt5a as a direct target of miR-539-5p. MiR-539-5p inhibited the expression of the Wnt5a and its downstream signaling molecules including JNK, PKC and CaMKII, which played a critical role in regulating migration of rMSCs. Taken together, our results demonstrate that miR-539-5p negatively regulates migration of rMSCs induced by BHD through targeting Wnt5a. These findings provide evidence that miR-539-5p should be considered as an important candidate target for the development of preventive or therapeutic approaches against bone nonunions.

Huo Xue Tong Luo capsule ameliorates osteonecrosis of femoral head through inhibiting lncRNA-Miat.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine has a long history of treating various bone diseases including osteoporosis and osteonecrosis etc. In clinical treatment, Huo Xue Tong Luo capsule (HXTL capsule) containing Peach kernel, Safflower carthamus, Angelica sinensis, Ligusticum wallichii etc, is one of the mostly used prescriptions for treating osteonecrosis of the femoral head (ONFH) with promising effects. OBJECTIVES: This study aims to identify the underlying molecular mechanism of how HXTL capsule exerts its function to ameliorate ONFH. MATERIALS AND METHODS: All femoral bone tissues were collected during surgeries. Rat bone marrow mesenchymal stem cells (rMSCs) were used. Quantitative real time PCR was used to check the relative expression levels of genes. ChIP assay was performed to evaluate the binding of H3K4me3 and H3K27me3 in Miat promoter. RESULTS: We showed that HXTL capsule promoted osteogenesis in rat MSCs as demonstrated by quantitative real time PCR and Alizarin Red S staining. Then we found silencing the endogenous lncRNA-Miat could promote osteogenesis of rMSCs. In addition, the ChIP assay showed that HXTL capsule significantly increased occupancy of H3K27me3 and decreased H3K4me3 in promoter regions of Miat, meaning HXTL capsule inhibited Miat expression through histone modifications. At last, by examining the femoral heads samples obtained from patients with ONFH during total hip arthroplasty surgery, we found the RNA level of hMiat in necrotic tissue was much higher than that of normal tissue. CONCLUSIONS: Taken together, our study shows that lncRNA-Miat might play an important role in pathogenesis of ONFH, and HXTL capsule can promote osteogenesis to ameliorate ONFH through inhibiting the transcriptional expression of Miat, at least partially.

Amentoflavone enhances osteogenesis of human mesenchymal stem cells through JNK and p38 MAPK pathways.

Amentoflavone is a bioflavonoid found in a variety of traditional Chinese medicines such as Gingko and Selaginella tamariscina. It has been reported that amentoflavone has anti-inflammatory, antioxidant, antiviral and anticancer effects. However, the effect of amentoflavone on osteogenic differentiation of human mesenchymal stem cells (hMSCs) has not been studied. In this study, we aim to explore the effect of amentoflavone on the proliferation and osteogenic differentiation of hMSCs. The results showed that amentoflavone significantly enhanced the proliferation, alkaline phosphatase (ALP) activity and mineralization in hMSCs. Western blot analysis revealed that the expression of runt-related transcription factor 2 and osterix proteins was upregulated in amentoflavone-treated hMSCs. Furthermore, we investigated the possible signaling pathways responsible for osteogenic differentiation of hMSCs by amentoflavone. We found that amentoflavone significantly increased the levels of phosphorylated JNK and p-p38. The amentoflavone-induced increases of ALP and mineralization were significantly diminished when the JNK and p38 MAPK pathways were blocked by selected inhibitors (SP600125, SB203580) in hMSCs. Furthermore, in vivo evidence indicated that amentoflavone protected against the dexamethasone-induced inhibition of osteoblast differentiation in tg(sp7:egfp) zebrafish larvae. Thus, we showed for the first time that amentoflavone improves the osteogenesis of hMSCs through the JNK and p38 MAPK pathway. Amentoflavone may be beneficial in treating bone-related disorders.

[Study and evaluation of preparation of silybin PLGA microspheres by stainless steel membrane emulsification technique].

OBJECTIVE: The aim of the present study was to prepare uniform-sized silybin loaded poly (lactic-co-glycolic acid) (PLGA) microspheres in study of silybin with stainless steel membrane. METHOD: Silybin PLGA microspheres were prepared by stainless steel membrane emulsification. The preparation conditions were optimized by single-factor test and orthogonal experiment, and evaluating the mean diameters, the particle size distribution, drug loading, entrapment efficiency and morphology of microsphere. RESULT: Prepared microspheres were round and surface was smooth. The mean diameter was (4.961 +/- 0.56) microm. The span was (1.75 +/- 0.18). The entrapment efficiency was (54.997 +/- 4.05)% and the average drug loading was (23.6 +/- 1.70)%. CONCLUSION: The stainless steel membrane emulsification can be used to prepare the silybin PLGA microspheres. The mean diameters of the silybin PLGA microspheres can be controlled in certain level. Stainless steel membrane emulsification has great potentiality exploitation and utilization.

Salvianolic acid B promotes osteogenesis of human mesenchymal stem cells through activating ERK signaling pathway.

Salvianolic acid B, a major bioactive component of Chinese medicine herb, Salvia miltiorrhiza, is widely used for treatment of cardiovascular diseases. Our recent studies have shown that Salvianolic acid B can prevent development of osteoporosis. However, the underlying mechanisms are still not clarified clearly. In the present study, we aim to investigate the effects of Salvianolic acid B on viability and osteogenic differentiation of human mesenchymal stem cells (hMSCs). The results showed Salvianolic acid B (Sal B) had no obvious toxic effects on hMSCs, whereas Sal B supplementation (5µM) increased the alkaline phosphatase activity, osteopontin, Runx2 and osterix expression in hMSCs. Under osteogenic induction condition, Sal B (5µM) significantly promoted mineralization; and when the extracellular-signal-regulated kinases signaling (ERK) pathway was blocked, the anabolic effects of Sal B were diminished, indicating that Sal B promoted osteogenesis of hMSCs through activating ERK signaling pathway. The current study confirms that Sal B promotes osteogenesis of hMSCs with no cytotoxicity, and it may be used as a potential therapeutic agent for the management of osteoporosis.